The goal of this laboratory is to develop methods for stable introduction and expression of genes in human keratinocytes for gene therapy purposes. The in vivo approach directly introduces the gene into epidermis while in our ex vivo approach, we first isolate keratinocytes, insert the desired gene while in tissue culture, and graft the genetically modified keratinocytes back onto the donor. We have developed a novel in vivo approach for directly introducing and expressing genes in epidermis by injecting naked plasmid DNA into the dermis underlying the epidermis. The direct injection approach has been used to develop DNA vaccines for leishmaniasis. Direct injection of DNA expressing Leishmania surface proteins into mouse skin are able to induce a Th1 cell-mediated immune response to Leishmania and confer protection from Leishmania infection. Additionally, the co-injection of oligonucleotides containing immunostimulatory sequences (unmethylated CpG dinucleotides) with Leishmania lysate is also able to elicit a protective Th1 immune response. This suggests that CpG sequences present on plasmid DNA are responsible for eliciting the immune response seen with plasmid DNA vaccination. Another use of the direct injection approach is the expression of biological response modifiers such as cytokines and interferons. In a dog papilloma model, both plasmids expressing interferon-alpha and CpG immunostimulatory oligonucleotides are being assessed as a treatment for papillomas. Although the ex vivo approach can ensure that a much higher percentage of the keratinocytes contain the desired gene, long-term expression has been lacking. To achieve persistent long-term expression, we are using topical selection of keratinocytes containing a multi-drug resistance (MDR) selectable marker gene. Preliminary studies in skin raft cultures have been completed and demonstrate the feasibility of this approach. Topical selection for keratinocytes expressing MDR is now being assessed in vivo in animal models. To help achieve long term expression, studies have been initiated to characterize keratinocyte stem cells. The goal of these stem cell studies is to understand the biological behavior of keratinocyte stem cells and to identify a unique marker that can be used to purify these keratinocyte stem cells. Finally, we have characterized how a POU transcription factor, that is specifically expressed in epidermis, might influence and regulate epidermal differentiation. To determine the functional effects of this POU transcription factor, in vitro models of epidermal differentiation have been developed where keratinocytes are transduced with retroviral vectors containing the full-length and deletional mutants of the POU transcription factor. These studies demonstrate that overexpressing the POU factor in keratinocytes results in altered patterns of epidermal differentiation and increased proliferation, suggesting that this POU factor accelerates the differentiation process. In vivo functional studies utilizing transgenic models have also been initiated. The POU transcription factor is either being expressed in incorrect epidermal locations or mutated POU genes are expressed at the appropriate epidermal layers.